PROJECT SUMMARY/ABSTRACT The goal of precision oncology is to tailor present and future cancer therapies to specific patients based on the systematic genomic assessment of their tumors. Therefore, large-scale cancer genome sequencing efforts represent an important first step towards achieving this goal. Such studies have identified Kelch-like ECH Associated Protein 1 (KEAP1), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2, to be mutated in approximately 30% of all lung cancers. NRF2 is a transcription factor that regulates a network of genes that coordinate the cellular response to oxidative stress. Inactivating mutations in KEAP1 result in constitutive NRF2 activity and activation of the oxidative stress response pathway in cancer cells. Of note, 30% of lung adenocarcinoma patients carry oncogenic KRAS mutations. Of these KRAS mutant tumors, 20% display loss of function mutations in KEAP1, implicating the importance of the oxidative stress response pathway in initiation and/or maintenance in this tumor type. The high frequency of mutation of genes in this pathway makes it an attractive target for therapy in lung cancer and other cancer types. Objectives: This study will (1) define the role of Keap1 loss of function in vivo in the normal lung and in cancer development; (2) high-throughput identification of drug targets in Keap1-mutant cell lines utilizing a custom sgRNA library targeting ~5000 genes with known chemical inhibitors (3) test the requirement of top candidate genes for tumor maintenance in murine lung adenocarcinoma that may reveal new pathways and genes that converge with Kras and Keap1 function that can be pharmacologically targeted to treat cancer. Three specific aims are proposed to address these objectives: Aim 1: Determine the role of Keap1 loss of function mutations using in vivo A published lentiviral vector coupling Cre and Cas9 expression will be used to simultaneously initiate tumors and edit Keap1 in the lung epithelium of genetically engineered mice containing oncogenic Kras and p53 loss of function mutations. Changes in tumor burden and tumor grade will be analyzed. Aim 2: Identification of novel drug targets specific to Keap1-mutant tumor cells Synthetic genetic interactions in Keap1-mutant cells will be determined by high-throughput screening efforts utilizing an sgRNA library targeting ~5000 putative or bona-fide drug targets. These genetic interactions will be validated using various murine isogenic lung adenocarcinoma cell lines containing loss of function mutations in Keap1. Aim 3: Functional validation of putative Keap1-mutant synthetic lethal genes in vivo A conditional model of lung adenocarcinoma will be used to explore and validate the functional loss of synthetic lethal gene candidates in a Keap1-mutant specific fashion utilizing in vivo CRISPR gene editing.